A conserved glutamine plays a central role in LOV domain signal transmission and Its duration

Abigail I. Nash, Wen Huang Ko, Shannon M. Harper, Kevin H. Gardner

Research output: Contribution to journalArticle

67 Citations (Scopus)

Abstract

Light is a key stimulus for plant biological functions, several of which are controlled by light-activated kinases known as phototropins, a group of kinases that contain two light-sensing domains (LOV, light-oxygen-voltage domains) and a C-terminal serine/threonine kinase domain. The second sensory domain, LOV2, plays a key role in regulating kinase enzymatic activity via the photochemical formation of a covalent adduct between a LOV2 cysteine residue and an internally bound flavin mononucleotide (FMN) chromophore. Subsequent conformational changes in LOV2 lead to the unfolding of a peripheral Jα helix and, ultimately, phototropin kinase activation. To date, the mechanism coupling bond formation and helix dissociation has remained unclear. Previous studies found that a conserved glutamine residue [Q513 in the Avena sativa phototropin 1 LOV2 (AsLOV2) domain] switches its hydrogen bonding pattern with FMN upon light stimulation. Located in the immediate vicinity of the FMN binding site, this Gln residue is provided by the Iß strand that interacts with the Jα helix, suggesting a route for signal propagation from the core of the LOV domain to its peripheral Jα helix. To test whether Q513 plays a key role in tuning the photochemical and transduction properties of AsLOV2, we designed two point mutations, Q513L and Q513N, and monitored the effects on the chromophore and protein using a combination of UV-visible absorbance and circular dichroism spectroscopy, limited proteolysis, and solution NMR. The results show that these mutations significantly dampen the changes between the dark and lit state AsLOV2 structures, leaving the protein in a pseudodark state (Q513L) or a pseudolit state (Q513N). Further, both mutations changed the photochemical properties of this receptor, in particular the lifetime of the photoexcited signaling states. Together, these data establish that this residue plays a central role in both spectral tuning and signal propagation from the core of the LOV domain through the Iß strand to the peripheral Jα helix.

Original languageEnglish (US)
Pages (from-to)13842-13849
Number of pages8
JournalBiochemistry
Volume47
Issue number52
DOIs
StatePublished - Dec 30 2008

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Phototropins
Wave transmission
Glutamine
Oxygen
Light
Flavin Mononucleotide
Electric potential
Phosphotransferases
Chromophores
Tuning
Proteolysis
Circular dichroism spectroscopy
Mutation
Protein-Serine-Threonine Kinases
Hydrogen Bonding
Circular Dichroism
Point Mutation
Cysteine
Spectrum Analysis
Hydrogen bonds

ASJC Scopus subject areas

  • Biochemistry

Cite this

A conserved glutamine plays a central role in LOV domain signal transmission and Its duration. / Nash, Abigail I.; Ko, Wen Huang; Harper, Shannon M.; Gardner, Kevin H.

In: Biochemistry, Vol. 47, No. 52, 30.12.2008, p. 13842-13849.

Research output: Contribution to journalArticle

Nash, Abigail I. ; Ko, Wen Huang ; Harper, Shannon M. ; Gardner, Kevin H. / A conserved glutamine plays a central role in LOV domain signal transmission and Its duration. In: Biochemistry. 2008 ; Vol. 47, No. 52. pp. 13842-13849.
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abstract = "Light is a key stimulus for plant biological functions, several of which are controlled by light-activated kinases known as phototropins, a group of kinases that contain two light-sensing domains (LOV, light-oxygen-voltage domains) and a C-terminal serine/threonine kinase domain. The second sensory domain, LOV2, plays a key role in regulating kinase enzymatic activity via the photochemical formation of a covalent adduct between a LOV2 cysteine residue and an internally bound flavin mononucleotide (FMN) chromophore. Subsequent conformational changes in LOV2 lead to the unfolding of a peripheral Jα helix and, ultimately, phototropin kinase activation. To date, the mechanism coupling bond formation and helix dissociation has remained unclear. Previous studies found that a conserved glutamine residue [Q513 in the Avena sativa phototropin 1 LOV2 (AsLOV2) domain] switches its hydrogen bonding pattern with FMN upon light stimulation. Located in the immediate vicinity of the FMN binding site, this Gln residue is provided by the I{\ss} strand that interacts with the Jα helix, suggesting a route for signal propagation from the core of the LOV domain to its peripheral Jα helix. To test whether Q513 plays a key role in tuning the photochemical and transduction properties of AsLOV2, we designed two point mutations, Q513L and Q513N, and monitored the effects on the chromophore and protein using a combination of UV-visible absorbance and circular dichroism spectroscopy, limited proteolysis, and solution NMR. The results show that these mutations significantly dampen the changes between the dark and lit state AsLOV2 structures, leaving the protein in a pseudodark state (Q513L) or a pseudolit state (Q513N). Further, both mutations changed the photochemical properties of this receptor, in particular the lifetime of the photoexcited signaling states. Together, these data establish that this residue plays a central role in both spectral tuning and signal propagation from the core of the LOV domain through the I{\ss} strand to the peripheral Jα helix.",
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